The Study On The Mechanism Of TRPV1 Regulating Proliferation And Adipogenic Differentiation Of Adipose-Derived Stem Cells

Objective: Transient receptor potential vanilloid 1(TRPV1)is a hot target for clinical research on metabolic functional diseases of adipocytes such as obesity.It is expressed in both pre-adipocytes and adipocytes and is involved in the regulation of adipose differentiation and browning process.Adipose derived stem cells(ADSCs)as an important source of fat cells in the body,can differentiate into a variety of cells,it has become a hot seed cell in tissue engineering and regenerative medicine,because of its rich source and no ethical restrictions.Recently,it has been reported that TRPV1 is also expressed on human ADSCs,but its physiological significance has not been clarified.To further elucidate the effect of TRPV1 on obesity and to provide new research ideas for tissue engineering fat construction and reconstruction of defective soft tissues.In this study,the expression and changes of TRPV1 were determined on primary cultured rat ADSCs,and the effects of TRPV1 on ADSCs proliferation and white and brown adipogenic differentiation were investigated by using the classic TRPV1 agonist Capsaicin(Cap)and antagonist Capsazepine(CAPZ).Methods:(1)Rat V-ADSCs and S-ADSCs were cultured in the primary culture.The immunophenotype of ADSCs was identified by flow cytometry,and the functional indexes of Nanog,Oct4 and Sox2 were detected by immunofluorescence on the 3D cell spheres.Western blotting detected the expression of TRPV1 protein.(2)To explore the effects of Cap and CAPZ on ADSCs proliferation:(1)CCK-8 tested dose-effect(0.3 ?M,1 ?M,3 ?M,10 ?M,30 ?M,100 ?M)and time-effect(24,48,72h)of capsaicin(classic agonist for TRPV1,but desensitizes TRPV1 over time)on cellular activity for Visceral adipose derived stem cells(V-ADSCs)and Subcutaneous adipose derived stem cells(S-ADSCs).(2)The effects of Cap and CAPZ on ADSCs proliferation were compared in 2D and 3D cultures: The ADSCs and HUVECs were incubated with Cap(1 ?M,3 ?M,10 ?M)and CAPZ(1 ?M,3 ?M,10 ?M)at different concentrations,respectively.The changes in the number of living cells and the number of proliferative cells were detected by cck-8 and EdU,after 48h;The agarose microtemplate method was used to establish a stable ADSCs three-dimensional cell ball culture system,and the changes of cell ball diameter were observed after ADSCs were incubated by Cap(3 ?M)and CAPZ(3 ?M)for 48 h.(3)To explore the effect of TRPV1 on the brown adipogenic differentiation of SADSCs: Western blotting was used to detect the expression of TRPV1 during the brown adipogenic differentiation(0,2,5,8 day).Groups Con,Cap(1 ?M,3 ?M,10 ?M),Cap(3 ?M)+CAPZ(10 ?M),CAPZ(10 ?M)were set during the process of differentiation induced by brown lipid formation,the expression of brown lipid marker protein UCP1 was detected by Western blotting,and Nile red staining was used to analyze the lipid formation rate of neutral lipid.(4)To explore the effect of TRPV1 on the white adipogenic differentiation of VADSCs: Groups Con,Cap(1 ?M,3 ?M,10 ?M),Cap(3 ?M)+CAPZ(10 ?M),CAPZ(10 ?M)were set during the process of differentiation induced by white lipid formation,the expression of lipogenic transcription gene PPAR-? mRNA and protein were detected by q-PCR and Western blotting,and the lipid formation rate of neutral lipid was analyzed by Nile red staining.Results:(1)TRPV1 was expressed in both S-ADSCs and V-ADSCs of rats.Cap can promote proliferation of ADSCs from both sites.The detection results of CCK-8 showed that: 1,3,10(?M)Cap all enhanced the cell activity of ADSCs,among which 3 ?M capsaicin had the most significant effect.Treating visceral and subcutaneous ADSCs with 3 ?M Cap for different time,48 h and 72 h were obvious in proliferative effects.(2)CCK-8 and EdU showed that the TRPV1 antagonist CAPZ not only could not cancel the proliferation-promoting effect of Cap,but showed a more significant effect to promote the proliferation of ADSCs than that of Cap,by increasing the number of living cells and the number of EdU positive cells.HUVECs had no such proliferative effect.(3)Under 3D cultivation environment,Cap and CAPZ incubation for 48 h can both increase the diameter of ADSCs,among which CAPZ had a more significant effect.(4)Western blotting results showed that the expression of TRPV1 did not change significantly during the induction of brown adipogenic differentiation of S-ADSCs.Capsaicin can significantly promote the brown adipogenic differentiation of S-ADSCs,It up-regulated the expression of the thermogenic gene UCP1 protein,and increased the number of neutral lipid droplets in cells.CAPZ canceled the effect of capsaicin in promoting brown adipogenic differentiation.(5)Capsaicin can significantly promote the white lipid differentiation of VADSCs,which was manifested as dose-dependently increased the content of neutral lipid droplets in cells,and significantly up-regulated of the protein and mRNA levels of PPAR-?.CAPZ can block the effect of Cap and inhibit white lipid differentiation.Conclusion:(1)TRPV1 was expressed in subcutaneous and visceral ADSCs of rats;(2)In 2D and 3D conditions,CAPZ blocking TRPV1 or Cap desensitization can promote ADSC proliferation;(3)Cap activating TRPV1 can promote the brown adipogenic differentiation and the expression of UCP-1 in ADSCs;(4)Cap promoted white adipogenic differentiation of ADSCs,and its mechanism was related to the activation of TRPV1 up-regulating the lipogenic gene PPAR-?.